The discovery of catalytic RNA fundamentally changed the course of science. The subsequent realization that catalytic RNAs could be tailored to suit individual needs has been nothing less than inspiring. Indeed, the past ten years has seen the creative development of numerous RNA catalysts. Concurrently, the diversity of applications for these catalytic RNAs has been escalating. For example, catalytic RNAs are being developed for detection protocols, for therapeutic intervention of diseases, and for use as biochemical tools. As we continue to exploit the steadily increasing knowledge base of RNA structure, folding, and catalysis, designing and applying novel and effective RNA catalysts is becoming more and more tractable.
Ribozymes are RNA molecules having an enzymatic activity, which enables the ribozyme to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro. Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme, which is held in close proximity to an enzymatic portion of the RNA that acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA destroys its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
A catalytic RNA that can excise a specific RNA sequence out of a larger RNA (the trans-excision-splicing reaction), although not previously discovered or engineered, would be very useful as a biochemical tool and also as a potential new therapeutic strategy. For example, multiple turnover catalytic RNAs, or ribozymes, with this activity could be used to excise a disease-causing RNA region out of a native transcript, to remove a premature stop codon, or to restore a frameshift mutation, for example.